Microfluidic methods for non-thermal nucleic acid manipulations

• US 6,406,893 B1
• Filed: 11/20/2000
• Issued: 06/18/2002
• Est. Priority Date: 04/04/1997
• Status: Expired due to Term

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Technologies Corp (Revvity, Inc.)
• Inventors: Knapp, Michael, Parce, John Wallace, Kopf-Sill, Anne R., Bousse, Luc J.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/718,140
• Time in Patent Office: 575 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/287.2, 536/22.1, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33
• US Class Current: 435/91.2
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01L 2200/0605 Metering of fluids
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/18 Means for temperature control
• CPC Class Codes: B01L 2300/1833 using electrical currents in the sample itself
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0418 electro-osmotic flow [EOF]
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/0262 using touch-off at substrate or container
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: G01N 2035/1053 : for separating part of the liquid, e.g. filters, extraction phase
• CPC Class Codes: G01N 2035/1058 : for mixing
• CPC Class Codes: G01N 27/44791 : Microapparatus sample containers with integrated microfluidic structures B01L3/5027

Abstract

Integrated systems, apparatus, software, and methods for performing biochemical analysis, including DNA sequencing, genomic screening, purification of nucleic acids and other biological components and drug screening are provided. Microfluidic devices, systems and methods for using these devices and systems for performing a wide variety of fluid operations are provided. The devices and systems of are used in performing fluid operations which require a large number of iterative, successive or parallel fluid manipulations, in a microscale, or sealed and readily automated format.

Claims

1. A method of amplifying a target nucleic acid by a non-thermal polymerase chain reaction, the method comprising:

providing a microfluidic device which includes a reaction chamber or channel, one or more sources of a target nucleic acid and primer sequences, a source of a chemical denaturant and a source of polymerase enzyme fluidly connected to the reaction chamber or channel, and a fluid direction system for delivering the chemical denaturant or the polymerase enzyme to the reaction chamber or channel;

flowing the target nucleic acid and the primer sequences along the reaction chamber or channel;

melting complementary strands of the flowing target nucleic acid by delivering a volume of the chemical denaturant to the reaction chamber or channel;

annealing the primer sequences to the flowing target nucleic acid by eliminating a denaturing effect of the chemical denaturant;

extending the primer sequences along the flowing target nucleic acid by delivering a volume of the polymerase enzyme to the reaction chamber or channel; and

repeating the steps of melting, annealing and extending to amplify the flowing target nucleic acid.

2. The method of claim 1, wherein the chemical denaturant comprises a base, and annealing the primer sequences to the target nucleic acid comprises neutralizing the base by delivering an effective amount of an acid to the reaction chamber or channel.

3. The method of claim 1, wherein the base is NaOH and the acid is HCl.

4. The method of claim 1, wherein the polymerase enzyme is selected from the group consisting of E. coli DNA pol I, Klenow fragment, T7 DNA polymerase, Taq polymerase, Pfu DNAse, Bst and Vent polymerase.

5. The method of claim 1, wherein the steps of melting, annealing and extending are repeated from about 10 to about 50 times.

6. The method of claim 1, wherein providing the microfluidic device further comprises introducing the target nucleic acid or the primer sequences from an external source.

7. The method of claim 6, wherein introducing the target nucleic acid or the primer sequences from an external source comprises transferring the target nucleic acid or the primer sequences to the reaction chamber or channel by a capillary, an electropipettor, or a sipping technology.

8. The method of claim 6, wherein the external source comprises a reagent storage substrate, a microtiter plate or an array of oligonucleotides.

9. The method of claim 1, wherein the primer sequences comprise all possible 5 mers or 6 mers.

10. The method of claim 1, wherein the primer sequences comprise a subset of all possible 5 mers or 6 mers.

11. The method of claim 1, wherein the source of polymerase enzyme further comprises nucleoside triphosphates or deoxynucleoside triphosphates.

12. The method of claim 1, wherein the chemical denaturant comprises a chaotropic agent, and wherein annealing the primer sequences to the target nucleic acid comprises desalting an amplification reaction mixture comprising the target nucleic acid, the primer sequences, and the polymerase.

13. The method of claim 12, wherein the chaotropic agent comprises urea or formamide.

14. The method of claim 12, wherein desalting the amplification reaction mixture comprises passing the amplification reaction mixture through a chromatographic matrix.

15. The method of claim 14, wherein the chromatographic matrix comprises a gel exclusion matrix or a nucleic acid binding matrix.

16. The method of claim 12, wherein desalting the amplification mixture comprises electrophoresing the amplification reaction mixture.

17. The method of claim 1, further comprising:

detecting an amplified target nucleic acid.

18. The method of claim 17, wherein detecting the amplified target nucleic acid comprises analyzing an amplification reaction mixture using a spectrophotometer, a fluorescent detector, a microscope, a fluorescent microscope, a scintillation counting device, or a combination thereof.

19. The method of claim 17, wherein detecting the amplified target nucleic acid comprises performing differentially labeled competitive probes hybridizations.

20. The method of claim 17, wherein detecting the amplified target nucleic acid comprises hybridizing one or more molecular beacons to the amplified target nucleic acid.

21. The method of claim 17, further comprising sequencing the amplified target sequence.

22. A method of producing an amplified nucleic acid in a microfluidic device, the method comprising:

providing a microfluidic device comprising a microscale channel;

flowing a double stranded nucleic acid in the microscale channel;

flowing a denaturant into the microscale channel and contacting the flowing double stranded nucleic acid, thereby providing a flowing denatured nucleic acid;

removing the denaturant; and

amplifying the flowing denatured nucleic acid to produce an amplified nucleic acid.

23. The method of claim 22, wherein flowing the double stranded nucleic acid further comprises introducing the double stranded nucleic acid from an external source.

24. The method of claim 23, wherein introducing the double stranded nucleic acid from an external source comprises transferring the double stranded nucleic acid to the microscale channel by a capillary, an electropipettor, or a sipping technology.

25. The method of claim 23, wherein the external source comprises a reagent storage substrate, a microtiter plate or an array of oligonucleotides.

26. The method of claim 22, wherein the denaturant comprises a base or a chaotropic agent.

27. The method of claim 22, wherein the denaturant is selected from the group consisting of NaOH, urea, and formamide.

28. The method of claim 22, wherein removing the denaturant comprises diluting the denaturant.

29. The method of claim 22, wherein the denaturant comprises a base and wherein removing the denaturant comprises providing an acid.

30. The method of claim 22, further comprising detecting the denatured nucleic acid or the amplified nucleic acid.

31. The method of claim 30, wherein detecting the denatured nucleic acid or the amplified nucleic acid comprises analyzing the nucleic acid using a spectrophotometer, a fluorescent detector, a microscope, a fluorescent microscope, a scintillation counting device, or a combination thereof.

32. The method of claim 30, wherein detecting the denatured nucleic acid or the amplified nucleic acid comprises performing differentially labeled competitive probes hybridizations.

33. The method of claim 30, wherein detecting the denatured nucleic acid or the amplified nucleic acid comprises hybridizing one or more molecular beacons to the nucleic acid.

34. The method of claim 22, further comprising sequencing the amplified nucleic acid.